Because many intracellular and intercellular processes are membrane mediated, there has been a great deal of research on the reconstitution of biological membranes as a method to study these processes. Since the original development of a procedure to form artificial planar phospholipid bilayer membranes (Mueller et al., 1962) and the demonstration of the fusion of vesicles that contain ion channels to planar membranes (Miller and Racker, 1976), studies have employed a variety of artificial membrane systems and various methods for studying functional molecules that are incorporated in or bound to biological membranes (Hoekstra and Duzgunes, 1993). Some researchers have incorporated biotinylated lipids (e.g., see Wright and Huang, 1992) or functional enzymes (e.g., see Hianik et al., 1996) into artificial membranes, but the purpose has been to study properties of the membranes or the incorporated molecules, not to use the synthetic membranes as a tool to study the properties of exogenous molecules as in the present invention. For a review of current techniques and research in this area (see Ottova and Tien, 1997).
Phospholipase C (PLC) is a generic name for enzymes that catalyze the hydrolysis of phosphoglycerides into diacylglycerols and phosphorylated alcohols such as serine, choline, inositol, glycerol, or ethanolamine. For example, a specific phospholipase C hydrolyzes phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4,5-bisphosphate (PIP.sub.2), resulting in each case in the formation of two second messengers: a hydrophobic diacylglycerol and a hydrophilic inositol phosphate (IP.sub.2 or IP.sub.3 respectively). This hydrolysis can be monitored by a variety of methods using endogenously or exogenously labeled substrates.
De Vivo (1994) describes current methods used for measuring the hydrolysis of PIP.sub.2. In the endogenous substrate approach, the cells of interest are cultured in the presence-[.sup.3 H]-inositol. The cells convert inositol to phosphatidylinositol (PI) using phosphatidylinositol synthase, and the PI in turn is converted to PIP-[.sup.3 H] and PIP.sub.2 -[.sup.3 H] by phosphatidylinositol kinases. When the polyphosphoinositide pool is labeled to a steady state, the breakdown of PIP and PIP.sub.2 is initiated by the addition to the cell culture medium of an appropriate stimulatory factor (e.g., receptor agonists for intact cells or guanylyl nucleotides for permeabilized cells).
In the exogenous substrate approach, purified labeled phospholipids are used as the substrate. The phospholipids are mixed in the presence or absence of detergent and sonicated briefly on ice to prepare vesicles. Aliquots of the substrate are mixed with a source of PLC (membranes or purified enzyme) and, often, G-protein subunits.
In both the endogenous and exogenous substrate approaches, solvent extraction is used to separate the hydrophilic reaction products from the hydrophobic substrate. Current methods for sphingomyelinase assays also require solvent extraction steps. It would be desirable to eliminate the extraction step for environmental and health reasons. In addition, it is difficult to automate the extraction of large numbers of samples, as would be necessary for example in high throughput screening of drug candidates.